Accuracy of genotype imputation based on random and selected reference sets in purebred and crossbred sheep populations and its effect on accuracy of genomic prediction.

BACKGROUND: The objectives of this study were to investigate the accuracy of genotype imputation from low (12k) to medium (50k Illumina-Ovine) SNP (single nucleotide polymorphism) densities in purebred and crossbred Merino sheep based on a random or selected reference set and to evaluate the impact of using imputed genotypes on accuracy of genomic prediction. METHODS: Imputation validation sets were composed of random purebred or crossbred Merinos, while imputation reference sets were of variable sizes and included random purebred or crossbred Merinos or a group of animals that were selected based on high genetic relatedness to animals in the validation set. The Beagle software program was used for imputation and accuracy of imputation was assessed based on the Pearson correlation coefficient between observed and imputed genotypes. Genomic evaluation was performed based on genomic best linear unbiased prediction and its accuracy was evaluated as the Pearson correlation coefficient between genomic estimated breeding values using either observed (12k/50k) or imputed genotypes with varying levels of imputation accuracy and accurate estimated breeding values based on progeny-tests. RESULTS: Imputation accuracy increased as the size of the reference set increased. However, accuracy was higher for purebred Merinos that were imputed from other purebred Merinos (on average 0.90 to 0.95 based on 1000 to 3000 animals) than from crossbred Merinos (0.78 to 0.87 based on 1000 to 3000 animals) or from non-Merino purebreds (on average 0.50). The imputation accuracy for crossbred Merinos based on 1000 to 3000 other crossbred Merino ranged from 0.86 to 0.88. Considerably higher imputation accuracy was observed when a selected reference set with a high genetic relationship to target animals was used vs. a random reference set of the same size (0.96 vs. 0.88, respectively). Accuracy of genomic prediction based on 50k genotypes imputed with high accuracy (0.88 to 0.99) decreased only slightly (0.0 to 0.67% across traits) compared to using observed 50k genotypes. Accuracy of genomic prediction based on observed 12k genotypes was higher than accuracy based on lowly accurate (0.62 to 0.86) imputed 50k genotypes.

Fibroblast growth factor receptor-1 mediates chemotaxis independently of direct SH2-domain protein binding.

Endothelial cells expressing fibroblast growth factor receptor-1 (FGFR-1) migrate and proliferate in response to treatment with FGF. We analysed ligand-induced migration and proliferation of porcine aortic endothelial cells expressing wild-type FGFR-1, point-mutated Y766F FGFR-1, unable to activate phospholipase C-gamma1 (PLC-gamma1), or carboxyl-terminally truncated FGFR-1, lacking either 48 (from amino acid 774 in the FGFR-1 sequence) or 63 (from amino acid 759) amino acid residues of the C-terminal tail. The truncated CT63 FGFR-1 mutant failed to mediate chemotaxis, but was in response to ligand stimulation capable of mediating proliferation of the cells, stimulation of MAP kinase activity and tyrosine phosphorylation of FRS2, an FGFR-1 specific signaling molecule. The defect in migration-capacity of CT63 was not due to loss of Y766, and thereby PLC-gamma1 activation, since cells expressing the mutant Y766F FGFR-1 migrated as efficiently as the wild-type receptor cells. Induction of phospholipase A2 (PLA2) activity by the activated FGFR-1 was dependent on the presence of Y766, and was therefore also not critical for the chemotactic response. Although the FGFR-1 only very inefficiently mediates activation of phosphatidylinositol 3' kinase (PI 3-kinase), the PI 3-kinase inhibitor wortmannin suppressed wild-type FGFR-1 mediated migration. We conclude that the signal transduction pathway for FGFR-1 mediated migration is independent of phosphotyrosine residues in the receptor and requires activation of a wortmannin-sensitive enzyme.

Contribution of Src and Ras pathways in FGF-2 induced endothelial cell differentiation.

We have examined fibroblast growth factor (FGF) receptor-1 mediated signal transduction in differentiation of endothelial cells (EC). The activated FGFR-1 couples to Ras through two adaptor proteins, FRS2 and Shc. In FGF-2 treated proliferating EC, FRS2 as well as Shc are tyrosine phosphorylated and interact with Grb2. In contrast, in FGF-2 treated differentiating cells, Shc, but not FRS2, is engaged in Grb2-interactions. Sustained MAP kinase activity has previously been implicated in differentiation. In FGF stimulated proliferating and differentiating endothelial cells, the MAP kinase Erk2 is activated in a sustained manner. Inhibition of MEK and MAP kinase activity by PD98059 treatment of cells, still allows EC tube formation. The FGFR-1 mediates activation of protein kinase C (PKC) through direct binding and activation of phospholipase C-gamma (PLC-gamma), and has also been shown to activate the cytoplasmic tyrosine kinase Src. Treatment of the cells with the PKC inhibitor bisindolylmaleimide does not prevent tube formation. In contrast, Src kinase activity is a prerequisite for EC differentiation, since treatment of the cells with PP1, a Src family specific inhibitor, abrogates tube formation. In differentiating EC, FGF-2 induces complex formation between Src and focal adhesion kinase (FAK). These data indicate that the Ras pathway is initiated via Shc or FRS2, dependent on the cellular program. Blocking the function of Src family kinases, attenuates differentiation.

Signal transduction by fibroblast growth factor receptors.

The fibroblast growth factor family, with its prototype members acidic FGF (FGF-1) and basic FGF (FGF-2), binds to four related receptor tyrosine kinases, expressed on most types of cells in tissue culture. In many respects, the FGF receptors appear similar to other growth factor receptors. Thus, dimerization of receptor monomers upon ligand binding is likely to be a requisite for activation of the kinase domains, leading to receptor trans phosphorylation. FGF receptor-1 (FGFR-1), which shows the broadest expression pattern of the four FGF receptors contains at least seven tyrosine phosphorylation sites. A number of signal transduction molecules are affected by binding with different affinities to these phosphorylation sites. The potential roles of these signal transduction molecules in FGF-induced biological responses and in pathological processes are discussed.

Identification of a chicken homologue in the Brn-3 subfamily of POU-transcription factors.

Among the many transcription factors thus far identified several are found to be expressed almost exclusively in the nervous system. The Brn-3 subfamily of POU-transcription factors constitutes a highly conserved group of such factors showing expression predominantly in sensory neurons. We now describe the nucleotide sequence and proposed amino acid sequence of a chicken homologue to the murine and human Brn-3 genes. Furthermore we characterise the early embryonic expression pattern of this chicken Brn-3 gene and show it to be expressed in peripheral sensory ganglia as well as in retinal ganglion cells. Based on these findings we conclude that the chicken homologue to the murine and human Brn-3a genes has been cloned. We have begun to examine possible regulatory pathways of Brn-3a by stimulating chick embryonic peripheral ganglia with trophic factors and assaying resulting levels of Brn-3a with a quantitative PCR approach. Trigeminal and dorsal root ganglia stimulated in culture by NGF and NT-3 embryonic day 9 (E9) produce neurites without raising the Brn-3a mRNA levels.

Shc and a novel 89-kDa component couple to the Grb2-Sos complex in fibroblast growth factor-2-stimulated cells.

A major pathway for mitogenicity is gated via the small GTP-binding protein Ras. Receptor tyrosine kinases couple to Ras through the Src homology 2 (SH2) domain protein Grb2. The activated fibroblast growth factor receptor-1 (FGFR-1) expressed in L6 myoblasts did not bind Grb2 directly, but indirectly, through the small adaptor protein Shc, which was tyrosine-phosphorylated in response to fibroblast growth factor-2 (FGF-2) stimulation. A FGFR-1 mutant in which Tyr766, a known autophosphorylation site, was changed to Phe, mediated less efficient tyrosine phosphorylation of Shc. FGF-2 stimulation of mutant FGFR-1-expressing cells still allowed formation of complexes containing Shc, Grb2, and the nucleotide exchange factor Sos and mediation of a mitogenic signal. Another pool of Grb2 was found in complex with a tyrosine-phosphorylated 89-kDa component after FGF-2 stimulation. Stimulation with other growth factors did not lead to tyrosine phosphorylation of p89. As shown by "far-Western" analysis, p89 bound directly to the Grb2 SH2 domain, and this interaction was inhibited by a peptide containing the Y(P)-X-N motif. Tyrosine-phosphorylated p89 was found exclusively in the membrane fraction, indicating its role in bringing Grb2, as well as Sos, to the plasma membrane. These data support the concept of growth factor-specific coupling of Grb2 to the Ras pathway.

Association of a 85-kDa serine kinase with activated fibroblast growth factor receptor-4.

Fibroblast growth factors (FGFs) transduce a variety of biological signals via four distinct tyrosine kinase receptors. We have characterized the phosphorylation of FGF receptor 4 (FGFR-4) and its association with a putative substrate, p85, using transfected L6 myoblast and NIH3T3 fibroblast cell lines. FGFR-4 was phosphorylated in vivo and in vitro mainly on serine and threonine residues in several peptides and to a lower degree on tyrosine residues. When analyzed further by in-gel kinase assay, immunoprecipitates of ligand-activated FGFR-4 contained a serine autophosphorylated polypeptide doublet of 85 kDa. Analysis of the major autophosphorylation site Y754F mutant of FGFR-4 showed that binding of p85 and its serine phosphorylation were independent of receptor autophosphorylation at this site. Okadaic acid treatment increased the basal autophosphorylation activity of p85 but decreased FGFR-4 tyrosine phosphorylation. In contrast, orthovanadate treatment increased the tyrosine phosphorylation of FGFR-4. These data show that a serine kinase is associated with activated FGFR-4 and suggest a role for serine phosphorylation in FGFR-4 function.

Fibroblast growth factor receptor-1-mediated endothelial cell proliferation is dependent on the Src homology (SH) 2/SH3 domain-containing adaptor protein Crk.

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) expressed on endothelial cells leads to cellular migration and proliferation. We have examined the role of the Src homology (SH) 2/SH3 domain-containing adaptor protein Crk in these processes. Transient tyrosine phosphorylation of Crk in fibroblast growth factor-2-stimulated endothelial cells was dependent on the juxtamembrane tyrosine residue 463 in FGFR-1, and a Crk SH2 domain precipitated FGFR-1 via phosphorylated Tyr-463, indicating direct complex formation between Crk and FGFR-1. Furthermore, Crk SH2 and SH3 domains formed ligand-independent complexes with Shc, C3G, and the Crk-associated substrate (Cas). Tyrosine phosphorylation of C3G and Cas increased as a consequence of growth factor treatment. We examined the role of Crk in FGFR-1-mediated cellular responses by use of cells expressing chimeric platelet-derived growth factor receptor-alpha/FGFR-1 (alphaR/FR) wild type and mutant Y463F receptors. The kinase activity of alphaR/FR Y463F was intact, but both Crk and the adaptor FRS-2 were no longer tyrosine-phosphorylated in the mutant cells. Both wild type and mutant receptor cells migrated efficiently, whereas cells expressing the mutant alphaR/FR Y463F failed to proliferate and Erk2 and Jun kinase activities were suppressed in these cells. In wild type alphaR/FR cells transiently expressing an SH2 domain mutant of Crk, Erk and Jun kinase activities as well as DNA synthesis were attenuated. Our data indicate that Crk participates in signaling complexes downstream of FGFR-1, which propagate mitogenic signals.

Signaling via fibroblast growth factor receptor-1 is dependent on extracellular matrix in capillary endothelial cell differentiation.

Differentiation of endothelial cells, i.e., formation of a vessel lumen, is a prerequisite for angiogenesis. The underlying molecular mechanisms are ill defined. We have studied a brain capillary endothelial cell line (IBEC) established from H-2Kb-tsA58 transgenic mice. These cells form hollow tubes in three-dimensional type I collagen gels in response to fibroblast growth factor-2 (FGF-2). Culture of IBEC on collagen gels in the presence of FGF-2 protected cells from apoptosis and allowed tube formation (i.e., differentiation) but not growth of the cells. FGF-induced differentiation, but not cell survival, was inhibited by treatment of the cells with an anti-beta1-integrin IgG. Changes in integrin expression in the collagen-gel cultures could not be detected. Rather, cell-matrix interactions critical for endothelial cell differentiation were created during the culture, as indicated by the gradual increase in tyrosine phosphorylation of focal adhesion kinase in the collagen-gel cultures. Inclusion of laminin in the collagen gels led to FGF-2-independent formation of tube structures, but cells were not protected from apoptosis. These data indicate that FGF receptor-1 signal transduction in this cell model results in cell survival. Through mechanisms dependent on cell-matrix interactions, possibly involving the alpha3beta1-integrin and laminin produced by the collagen-cultured IBE cells, FGF stimulation also leads to differentiation of the cells.

Role of thrombospondin-1-derived peptide, 4N1K, in FGF-2-induced angiogenesis.

Angiogenesis involves proliferation of capillary endothelial cells and formation of lumen-containing tube-like structures. A recently established murine brain capillary endothelial cell line, IBE, can either proliferate or form tube-like structures (i.e., differentiate) in response to fibroblast growth factor-2 (FGF-2), dependent on the culture conditions. The 4N1K peptide (KRFYVVMWKK), which is derived from the C-terminal cell-binding domain of thrombospondin-1 (TSP-1), inhibited tube formation, but not proliferation of IBE cells. Polyclonal antibodies against 4N1K blocked TSP-1-induced inhibition of tube formation by IBE cells. 4N1K inhibited tyrosine phosphorylation of focal adhesion kinase and FGF-2-stimulated tyrosine phosphorylation of phospholipase C-gamma in tube-forming, but not proliferating, IBE cells. The peptide also inhibited FGF-2-induced neovascularization in mouse cornea. Our results indicate that TSP-1 may exert its inhibitory effects on angiogenesis via the C-terminal cell-binding domain containing the 4N1K sequence by inhibiting tube formation by endothelial cells.

The PI 3-kinase isoforms p110(alpha) and p110(beta) have differential roles in PDGF- and insulin-mediated signaling.

Phosphoinositide 3'-kinases constitute a family of lipid kinases implicated in signal transduction through tyrosine kinase receptors and heterotrimeric G protein-linked receptors. Phosphoinositide 3'-kinases that bind to the platelet-derived growth factor receptor are composed of two subunits: the p85 subunit acts as an adapter and couples the catalytic p110 subunit to the activated receptor. There are different isoforms of p85 as well as of p110, the individual roles of which have been elusive. Using microinjection of inhibitory antibodies specific for either p110(alpha) or p110(beta) we have investigated the involvement of the two p110 isoforms in platelet-derived growth factor- and insulin-induced actin reorganization in porcine aortic endothelial cells. We have found that antibodies against p110(alpha), but not antibodies against p110(beta), inhibit platelet-derived growth factor-stimulated actin reorganization, whereas the reverse is true for inhibition of insulin-induced actin reorganization. These data indicate that the two phosphoinositide 3'-kinase isoforms have distinct roles in signal transduction pathways induced by platelet-derived growth factor and insulin.

Suc1-associated neurotrophic factor target (SNT) protein is a major FGF-stimulated tyrosine phosphorylated 90-kDa protein which binds to the SH2 domain of GRB2.

In Swiss 3T3 fibroblasts, basic fibroblast growth factor (bFGF) stimulates the unique tyrosine phosphorylation of a protein complex around 90 kDa, as ascertained by high resolution 2-D PAGE and anti-phosphotyrosine blotting. The majority of this complex consists of the protein(s) designated previously as SNT (suc1-associated neurotrophic factor target). Tyrosine phosphorylated SNT binds to both p13suc1 protein and the SH2 domain of Grb2. Binding of SNT to Grb2 is likely to be mediated through the consensus binding motif, pYXN, on SNT. The binding of SNT to p13suc1 is independent of the pYXN motif. Tyrosine phosphorylated SNT is localised in the plasma membrane where it could form a complex with Grb2 and Sos, enabling the initiation of a novel FGF-specific signalling pathway.